Indel marker for identifying sex of larimichthys crocea in different habitats and application thereof

Abstract

The invention discloses an indel marker for identifying sex of larimichthys crocea in different habitats and application thereof, the indel marker is an insertion / deletion nucleotide sequence with the length of 12bp at the 47617128-site basic group of the No.3 chromosome of the larimichthys crocea, the sequence is as shown in SEQ ID NO.1, a female individual has the sequence as shown in SEQ ID NO.1, and a male individual deletes the sequence as shown in SEQ ID NO.1. The invention also discloses a primer and a kit for amplifying the marker, a method for identifying the sex of the large yellow croaker in different habitats by adopting the primer or the kit, and application of the primer, the kit or the method in identifying the sex of the large yellow croaker in different habitats. The method has the characteristics of universality, high resolution, specificity, low cost, easiness in operation and the like, and also has industrialization potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and particularly relates to an indel marker for identifying the gender of different habitats of Pseudosciaena crocea and application thereof. BACKGROUND

[0002] Most fish lack heteromorphic sex chromosomes, and the sex determination region is difficult to directly identify at morphological and molecular levels. This feature makes it impossible to accurately identify the gender of Pseudosciaena crocea in vivo by traditional morphological methods, especially in the juvenile stage (gonad undifferentiation period). There is a significant difference in growth between males and females in Pseudosciaena crocea in aquaculture production, which makes monosex culture have great industrial value. The development of gender-specific molecular markers provides a technical basis for realizing monosex breeding of Pseudosciaena crocea and analyzing the evolution and domestication selection of sex determination genes.

[0003] A gender-specific deletion marker between dmrt1 and cfap157 genes has been identified in the Daiqu population. Verification of this marker in 276 Pseudosciaena crocea of the Daiqu population showed 100% accuracy, but the identification coincidence rate in the Minyue East population was only 79.6%, indicating that the sex determination region is differentiated from the Minyue East population. A perfect gender-linked SNP has been screened by whole-genome scanning of Pseudosciaena crocea in the Minyue East population. Based on this SNP, an allele-specific PCR (AS-PCR) detection system was developed (using two universal primers and one Y chromosome-specific primer, only a 348 bp band is amplified in female fish, while an additional 194 bp Y chromosome-specific band is produced in male fish). This marker was verified in nearly 2200 individuals of the Minyue East and Daiqu populations, with an accuracy rate of 100%. Further positioning analysis showed that the sex determination region of the Minyue East population is located on chromosome 22, which is in sharp contrast to the positioning of chromosome 3 in the Daiqu population, revealing the genetic differentiation of sex determination mechanisms between populations. It is worth noting that although this marker is also effective in the Daiqu population, a deletion marker specific to the Daiqu population can be more precisely adapted to the genetic background of this population. The study of the sex determination mechanism of Pseudosciaena crocea in the Naozhou population is relatively lagging behind. Due to long-term wild state and lack of domestication, the basic biology of the Naozhou population started late, and targeted gender-linked marker studies have not been reported.

[0004] In addition to geographical population differences, genetic and morphological differentiation between wild and farmed populations (different ecological environments, referred to as habitats) also influences the application of sex markers. There are differences in genetic diversity between wild and farmed populations. Studies based on mitochondrial D-loop region analysis and microsatellite markers have shown that the haplotype diversity index, observed heterozygosity, and Shannon index of wild populations are higher than those of farmed populations. Other studies have shown differences in sex dimorphism expression; in wild populations, female body length and male body height have the greatest direct effect on body weight; while in farmed populations, the total length of both male and female fish has the most significant effect on body weight. Therefore, it is necessary to develop an indel marker for sex in large yellow croaker from different habitats. Summary of the Invention

[0005] The purpose of this invention is to provide an indel marker for identifying the sex of large yellow croaker from different habitats.

[0006] The present invention also aims to provide a primer for amplifying the indel marker and a kit including the primer, as well as a method for identifying the sex of large yellow croaker from different habitats using the primer or kit.

[0007] The final object of the present invention is to provide the application of the above-described primers, kits or methods in identifying the sex of large yellow croaker in different habitats.

[0008] The first objective of the present invention can be achieved by the following technical solution: an indel marker for identifying the sex of large yellow croaker from different habitats, wherein the indel marker is a 12bp insertion / deletion nucleotide sequence at position 47617128 of chromosome 3 of large yellow croaker, the sequence being as shown in SEQ ID NO.1, with female individuals having the sequence shown in SEQ ID NO.1 and male individuals lacking the sequence shown in SEQ ID NO.1.

[0009] Based on high-throughput resequencing data, this invention screens sex-associated SNPs and InDel molecular markers from wild and farmed populations of large yellow croaker, as well as from the Naozhou and Eastern Fujian-Guangdong populations. Combining population genetics analysis, GWAS, and FST methods, stable sex-associated loci are identified, and specific primers are designed to achieve efficient sex identification. Furthermore, a universal sex molecular marker for large yellow croaker applicable to wild populations, farmed populations, and different geographical subpopulations is developed.

[0010] The development process of sex indel markers for large yellow croaker in different habitats in this invention includes: sample collection → morphological feature analysis → resequencing and SNP / Indel mining → GWAS analysis → sex-associated indel marker screening → primer design and validation → molecular marker application.

[0011] in:

[0012] Preferably, sample collection includes:

[0013] A total of 146 samples (58 females and 88 males) of different large yellow croaker groups were collected. Among them, 20 wild large yellow croaker (NZ) samples (10 females and 10 males) were collected in the waters near Naozhou Island, Zhanjiang City, Guangdong Province (longitude: 110.51639°; latitude: 20.75070°); 68 wild large yellow croaker samples (22 females and 46 males) were collected in the waters off Liu'ao Town, Zhangpu City, Fujian Province (longitude: 117.70330°; latitude: 23.89065°); and 58 domesticated and farmed large yellow croaker samples (26 females and 32 males) were collected from Sandu Marine Food Co., Ltd., Ningde City, Fujian Province (longitude: 119.57384°; latitude: 26.60169°).

[0014] Preferably, the morphological characteristics analysis of male and female large yellow croaker includes:

[0015] Images were acquired from well-preserved large yellow croaker specimens. The specimens were fixed to foam boards, and the fins were spread out using pins to ensure natural extension without abnormal bending. Each specimen was then photographed individually. During photography, the camera was fixed so that the lens was completely perpendicular to the specimen's shooting surface, maintaining consistent specimen placement and focus. Distinctive and easily identifiable points on the large yellow croaker were selected as landmarks. Geometric morphology methods were used to perform Procrustes superposition and average morphological deformation analysis on both sexes. TPS interpolation was used to transform the difference between the two average shapes into a smooth, continuous deformation field, and the spatial distribution pattern of this difference across the entire morphology was visually visualized using a deformation regular grid.

[0016] Preferably, resequencing and SNP / Indel mining include DNA extraction and library preparation, high-throughput sequencing, quality control, alignment and variant detection, and PCA analysis.

[0017] Preferably, DNA extraction and library construction includes: extracting high-quality genomic DNA using the CTAB method and constructing an Illumina sequencing library.

[0018] Preferably, high-throughput sequencing includes 150bp paired-end sequencing on the Illumina NovaSeq platform.

[0019] Preferably, quality control includes: using FASTP to remove adapters and low-quality sequences from the raw sequencing data.

[0020] Preferably, the alignment and variant calling includes: aligning clean reads to the reference genome (reference genome version GCF_000972845.2) using BWA-MEM, followed by SNP and Indel calling using GATK.

[0021] Preferably, the PCA analysis includes:

[0022] Filtering the variant data using PLINK (e.g. MAF>0.05, missing rate<10%).

[0023] Perform principal component analysis with PCA.

[0024] Plot the principal component plot in R, color-coded by sex.

[0025] Preferably, the gender association analysis (GWAS) includes:

[0026] Perform gender phenotype-based association analysis using PLINK:

[0027] plink --bfile filtered_data --assoc --pheno sex.txt --allow-no-sex

[0028] Use Bonferroni correction for significance threshold.

[0029] Generate QQ plot and Manhattan plot (using qqman or CMplot R package).

[0030] Preferably, the FST analysis pipeline includes:

[0031] Significance threshold: determine FST significance threshold (FST>0.2) by permutation test (1000 times).

[0032] Preferably, the Indel marker development includes: screen Indel sites with length≥3bp within the gender association interval, and clear sex typing.

[0033] Preferably, the marker type includes: Indel-based codominant molecular marker, suitable for general PCR and electrophoresis platform. Detection efficiency: single pair of primers can achieve sex typing, accuracy≥98%.

[0034] The final selected indel marker is located at ri-3-47617128 (reference genome version GCF_000972845.2), which has a 12bp long insertion / deletion nucleotide sequence at this site, and the insertion / deletion nucleotide sequence is shown as SEQ ID NO. 1.

[0035] Specifically: 5'-TGCAGGCCGATG-3' (as shown in SEQ ID NO. 1).

[0036] The above second object of the present application can be achieved by the following technical solution: a primer for amplifying the indel marker, the primer comprising an upstream primer and a downstream primer, the sequence of the upstream primer being as shown in SEQ ID NO. 2, and the sequence of the downstream primer being as shown in SEQ ID NO. 3.

[0037] Specifically:

[0038] Forward: 5'-CCACCACTTCATTCCTGGACA-3' (as shown in SEQ ID NO. 2);

[0039] Reverse: 5'-CTCATTCCTCTGCTCCCTACAA-3' (as shown in SEQ ID NO. 3).

[0040] The present application also provides a kit for identifying the gender of large yellow croaker in different habitats, which comprises the primer.

[0041] The present application also provides a method for identifying the gender of large yellow croaker in different habitats, comprising the following steps:

[0042] (1) extracting the genomic DNA of the sample to be tested;

[0043] (2) using the primer or the kit to perform PCR amplification on the genomic DNA of the sample to be tested as a template;

[0044] (3) judging the gender of the large yellow croaker according to the electrophoretogram, and the individual only having a 131bp fragment is a female large yellow croaker, and the individual having both 131bp and 119bp fragments is a male large yellow croaker.

[0045] In the method for identifying the gender of large yellow croaker in different habitats:

[0046] Preferably, in the PCR amplification in step (2), the PCR reaction system used is: 2x Taq MasterMix 10 μL, 0.5 μL of the upstream and downstream primers each with a concentration of 10 μM, 50 ng of genomic DNA, and ddH2O to 20 μL.

[0047] Preferably, in the PCR amplification in step (2), the PCR reaction program used is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 9 s, 35 cycles, and 72℃ extension for 5 min.

[0048] Preferably, in step (3), female: single band (131 bp); male: double bands (131 bp and 119 bp).

[0049] The last purpose of the present application can be achieved by the following technical solutions: the application of the above-mentioned primer or the above-mentioned kit or the above-mentioned method in identifying the gender of large yellow croaker in different habitats.

[0050] The present application has the following advantages:

[0051] (1) Universality: suitable for gender identification of Naozhou population and Min-Yue East population of large yellow croaker, and also suitable for gender identification of wild and cultured large yellow croaker;

[0052] (2) High resolution and specificity: combined with GWAS and FST analysis, potential gender determination regions are determined, and Indel sites are accurately located;

[0053] (3) Low cost and easy operation: without complex equipment, ordinary laboratory can complete the detection through conventional PCR, and the cost of a single sample is less than 5 yuan;

[0054] (4) Industrialization potential: the developed molecular marker can be converted into a kit to promote monosex culture and genetic breeding of large yellow croaker. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 For morphological difference analysis of large yellow croaker in Example 1, Figure A is a geometric morphological landmark of large yellow croaker, Figure B shows a body deformation grid map of female and male large yellow croaker based on geometric morphological landmarks, the left graph in Figure B is the average morphological grid of female, the right graph in Figure B is the average morphological grid of male, and Figure C visualizes the relative deformation of female based on the reference benchmark of male average shape;

[0056] Figure 2 For a typical example of anatomical histological identification phenotype of large yellow croaker for resequencing and molecular marker verification in Example 2 and Example 5, Figure A is an ovary appearance graph of female large yellow croaker, Figure B is a sperm nest appearance graph of male large yellow croaker, Figure C is an ovary histological section graph, and the microstructure can be seen ovum cell, Figure D is a sperm nest histological section graph, and the microstructure can be seen spermatocyte and sperm cell;

[0057] Figure 3 For population structure and gender correlation analysis of female and male large yellow croaker in Example 2, Figure A is a principal component analysis (PC1 vs PC2) based on whole genome SNP, and the ellipse represents 95% confidence interval, Figure B is a comparison of observed value and theoretical expected value distribution of gender correlation analysis, Figure C is a whole genome Manhattan graph of gender correlation SNPs site, the abscissa is the chromosome position, and the ordinate is -log 10(P), the red line is the Bonferroni corrected significance threshold (P = 5 x 10 -8 ), the blue line is the suggestive threshold (P = 1 x 10 -5 ), the significant peak on chromosome 3, D is the Manhattan plot of gender-associated SNPs on chromosome 3 of P. tridentatus, each point represents a SNP site, the abscissa is its physical position on chromosome 3 (unit: Mb), and the ordinate is the logarithmic conversion of the association significance (-log 10 (p value)), the red dotted line represents the Bonferroni corrected significance threshold, and E is the local Manhattan plot of the 45-50 Mb interval on chromosome 3;

[0058] Figure 4 For the Fst analysis of male and female SNP data of P. tridentatus in Example 3, A is the whole genome FST distribution map between male and female P. tridentatus, each point in the figure represents the FST value of a SNP site, the abscissa is its physical position in the whole genome, and the ordinate is the FST value, different chromosomes are distinguished by different colors, B is the FST distribution map of SNPs on chromosome 3, each point represents the FST value of a single SNP on chromosome 3, the abscissa is the physical position (unit: Mb), and the ordinate is the FST value, C is the 1 Mb window average FST sliding map of chromosome 3;

[0059] Figure 5 For the population structure and gender correlation analysis of male and female P. tridentatus in Example 4, A is the whole genome Manhattan plot of gender-associated Indel sites, the abscissa is the chromosome position, and the ordinate is -log 10 (P), the red line is the Bonferroni corrected significance threshold (P = 5 x 10 -8 ), the blue line is the suggestive threshold (P = 1 x 10 -5 ), B is the Manhattan plot of gender-associated Indels on chromosome 3 of P. tridentatus, each point represents an INDEL site, the abscissa is its physical position on chromosome 3 (unit: Mb), and the ordinate is the logarithmic conversion of the association significance (-log 10 (p value)), the red dotted line represents the Bonferroni corrected significance threshold, C is the local Manhattan plot of the 45-50 Mb interval on chromosome 3, which shows the gender association signal in the 45-50 Mb interval on Chr3 in an enlarged manner;

[0060] Figure 6 For the electrophoretic typical results in Example 5. DETAILED DESCRIPTION

[0061] Unless otherwise specified, all reagents and materials used in the following examples are commercially available. Furthermore, unless otherwise specified, all laboratory instruments used are standard laboratory equipment.

[0062] Example 1: Morphological characteristics analysis of male and female large yellow croaker

[0063] Landmarks were selected from points on the large yellow croaker that are easily identifiable and have distinct characteristics. These were categorized into three types: Type I (intersections between different tissues), Type II (depressions or protrusions within the structure), and Type III (extreme points within the structure), as detailed below. Figure 1 As shown in Figure A, Procrustes superposition and average morphological deformation analysis were performed on both sexes using geometric morphology methods. The morphological deformation mesh results show that males and females are highly consistent in key morphological parameters such as body length ratio, dorsal and ventral contour, and head structure, with no obvious morphological deviations observed. Figure 1 (Figures B-C)

[0064] Specifically, the analysis of morphological differences in large yellow croaker is as follows: Figure 1 As shown, Figure A represents the geometric morphological markers of the large yellow croaker. Figure B shows the body deformation mesh diagrams of female and male large yellow croakers constructed based on the geometric morphological markers. The left image in Figure B is the average morphological mesh of the female, and the right image is the average morphological mesh of the male. The mesh deformation represents the morphological differences between the two sexes in the overall body shape. Stretched or contracted areas indicate morphological changes in the corresponding parts. The mesh shapes of the male and female are basically the same, and no obvious stretching or compression is shown, indicating that the male and female large yellow croakers do not show significant differences in overall body structure. Figure C visualizes the relative deformation of the female using the average shape of the male as a reference. The deformation diagram is constructed based on the superposition of Procrustes and morphological averaging. The deformation diagram shows that the arrows of most Landmarks are short and the changes are slight, indicating that there is no systematic shift in the local structure of the male and female body shapes, supporting the conclusion that the overall morphology of the two sexes is consistent.

[0065] Conclusion: Large yellow croaker did not exhibit morphological sex dimorphism between male and female individuals. Their body shape differences may be mainly driven by individual developmental stage or environmental factors, rather than sex itself.

[0066] Example 2: GWAS analysis based on SNP resequencing of male and female large yellow croaker

[0067] Anatomical sex confirmation

[0068] Target: All large yellow croaker individuals used for resequencing;

[0069] Operating steps:

[0070] Vivo-anesthesia (MS-222, 100 mg / L, immersion for 5 min);

[0071] Gonadal tissue was removed via ventral dissection;

[0072] Histological sections of gonads:

[0073] Fixation: Bouin's solution for 24 hours;

[0074] Sectioning: Paraffin sections were 5 μm thick;

[0075] Staining: HE staining;

[0076] The interpretation criteria are shown in Table 1 below:

[0077] Table 1 Gender Determination Criteria

[0078] Gender Microscopic features Illustrative examples Female Oocyte Figure 2 Figure 1A, 1C Male Spermatid structure Figure 2 Figures B and D

[0079] PCA analysis results ( Figure 3 (Figure A in the middle):

[0080] Principal component analysis results showed that male and female individuals exhibited significant separation along the PC1 and PC2 dimensions. Figure 3 Figure A shows that there are genetic differences between sexes. This provides a basis for subsequent detection of sex-related variations.

[0081] QQ image analysis ( Figure 3 (Figure B in the middle):

[0082] The QQ plot illustrates the distribution relationship between the observed p-values ​​and the expected p-values. The results show ( Figure 3 (Figure B) The vast majority of loci are distributed along the main diagonal, but deviations occur in extremely significant regions, suggesting the presence of variant loci highly associated with sex.

[0083] Manhattan diagram analysis ( Figure 3 (Figure C in the middle):

[0084] Manhattan plots reveal the presence of sex-related SNP sites on chromosome Chr3. Figure 3 (Figure C in the middle)

[0085] Partial Manhattan diagram ( Figure 3 (Diagrams D-E):

[0086] A significant correlation peak was observed at 46.5–49.5 Mb on chromosome 3. Figure 3 (See Figure D). A magnified view of the 45–50 Mb region of chromosome 3, showing the strongest sex-related correlation at 47.3–48.0 Mb. Figure 3 (China E diagram).

[0087] Specifically, the population structure and sex correlation analysis of large yellow croaker are as follows: CHRFigure 1. Genome-wide association study (GWAS) of sex-associated SNPs in P. olivaceus. A, Principal component analysis (PC1 vs PC2) based on whole-genome SNPs, female (red) and male (blue) individuals significantly separated on PC1, indicating gender-related genetic differentiation, ellipses represent: 95% confidence interval, B, Distribution of observed vs expected values of gender association analysis, slight deviation from the diagonal in the tail, suggesting the presence of real association signals, C, Genome-wide Manhattan plot of gender-associated SNPs sites, horizontal axis: chromosome position, vertical axis: -log 10 (P), red line: Bonferroni corrected significance threshold (P = 5 x 10 -8 ), blue line: suggested threshold (P = 1 x 10 -5 ), D, Manhattan plot of gender-associated snps on chromosome 3, each dot represents a SNP site, horizontal axis: its physical position on chromosome 3 (unit: Mb), vertical axis: log-transformed association significance (-log 10 (p-value)), red dotted line represents the significance threshold after Bonferroni correction, significantly associated sites are highly concentrated in the interval of about 46.5-49.5 Mb on Chr3, E, Local Manhattan plot of the interval 45-50 Mb on chromosome 3, zoom in to display the gender association signal in the interval 45-50 Mb on Chr3, part of the variation snps in this region show high linkage disequilibrium (LD), which may constitute a structure or regulatory region related to sex determination.

[0088] Information of SNPs based on GWAS screening through Bonferroni correction (BONF) significance threshold (P < 0.05) is shown (Table 2).

[0089] Table 2 SNPs based on GWAS screening through Bonferroni correction (BONF) significance threshold (P < 0.05)

[0090] SNP UNADJ GC BONF HOLM SIDAK_SS SIDAK_SD FDR_BH FDR_BY Figure 4 3 rs:3:47459569 1.485e-10 6.223e-09 0.001083 0.001083 0.001082 0.001082 0.0007442 0.01219 3 rs:3:47458183 2.041e-10 8.095e-09 0.001488 0.001488 0.001487 0.001487 0.0007442 0.01219 3 rs:3:47555780 1.462e-09 4.122e-08 0.01066 0.01066 0.01061 0.01061 0.003554 0.05822 3 rs:3:47823050 2.964e-09 7.394e-08 0.02162 0.02162 0.02139 0.02139 0.005405 0.08853 3 rs:3:47498003 5.721e-09 1.274e-07 0.04172 0.04172 0.04086 0.04086 0.006246 0.1023 3 rs:3:47506146 6.498e-09 1.415e-07 0.04739 0.04739 0.04628 0.04628 0.006246 0.1023 3 rs:3:47638843 6.5e-09 1.415e-07 0.04741 0.04741 0.0463 0.0463 0.006246 0.1023 3 rs:3:47638636 6.851e-09 1.478e-07 0.04997 0.04997 0.04874 0.04874 0.006246 0.1023

[0091] Note: CHR: Chromosome, Chromosome number; SNP: SNP Identifier, Variant site ID; UNADJ: Unadjusted P-value, Unadjusted raw P-value; GC: Genomic Control Adjusted P-value, Genomic control corrected P-value; BONF: Bonferroni Correction, Bonferroni corrected P; HOLM: Holm-Bonferroni Correction, Holm-Bonferroni corrected P-value; SIDAK_SS: Sidák Single Step Correction, Sidák single step corrected P-value; SIDAK_SD: Sidák Step-Down Correction, Sidák step-down corrected P-value; FDR_BH: Benjamini-Hochberg FDR, Benjamini-Hochberg FDR corrected; FDR_BY: Benjamini-Yekutieli FDR, Benjamini-Yekutieli FDR corrected.

[0092] Results of whole genome FST analysis

[0093] Results of whole genome FST analysis Figure 4 Figure 2A:

[0094] On a whole genome scale, the FST values of most SNPs are distributed between 0-0.1, indicating a low level of overall genomic differentiation between males and females. A high FST peak appears on chromosome 3, suggesting that this chromosome might harbor important genetic variations related to sex determination.

[0095] Results of FST analysis on chromosome 3 Figure 4 Figure 2B-C:

[0096] On Chr3, especially in the interval of 46.5-49.5 Mb, the FST values are significantly elevated. This extreme differentiation usually implies the presence of strong sexual selection pressure or sex determination sites. The 1 Mb sliding window average FST sliding window analysis on chromosome 3 helps to reduce the influence of random fluctuations and identify genomic regions with sustained high differentiation. The results show that the 46-50 Mb segment of chromosome 3 has a much higher average FST than other regions, further verifying that it is a significantly differentiated hotspot segment between males and females. This region is consistent with the results of the aforementioned GWAS and Manhattan plot analysis, providing cross-validation support.

[0097] Specifically, the Fst analysis of the male and female SNP data of P. olivaceus is as follows Figure 5Figure 1. Manhattan plot of genome-wide FST distribution between female and male P. major. Each point represents the FST value of a SNP locus. The horizontal axis represents the physical position of the SNP in the whole genome. The vertical axis represents the FST value. Different chromosomes are distinguished by different colors. Most of the SNP loci have low FST values, indicating low genetic differentiation between females and males. However, an abnormal increase in FST was observed in the region of chromosome 3, suggesting that this region may be related to sex determination. Figure 2. Manhattan plot of FST distribution of SNPs on chromosome 3. Each point represents the FST value of a single SNP on chromosome 3. The horizontal axis represents the physical position (unit: Mb). The vertical axis represents the FST value. There are multiple significantly high FST value loci in the 46-49 Mb interval, indicating significant differentiation between females and males in this region, which may be a candidate region for sex determination or regulation. Figure 3. 1 Mb window average FST sliding plot of chromosome 3. This plot shows the distribution of average FST values calculated by 1 Mb sliding window on chromosome 3. The horizontal axis represents the starting position of the sliding window. The vertical axis represents the average FST of SNPs in the window. A significantly higher average FST peak is observed in the 46-50 Mb interval.

[0098] Example 4 GWAS analysis based on Indel resequencing of female and male P. major

[0099] Manhattan plot analysis Figure 5 (A).

[0100] The Manhattan plot revealed the presence of Indel loci on chromosome 3 that were significantly associated with gender Figure 5 (A).

[0101] Local Manhattan plot Figure 5 (B)-(C).

[0102] A significant association peak was observed at 46.5-49.5 Mb on chromosome 3 Figure 5 (B). Enlarged view of the 45-50 Mb interval on chromosome 3 showing the strongest association with gender at 47.4-48.0 Mb CHR (C).

[0103] Information on Indels that passed the Bonferroni correction (BONF) significance threshold based on GWAS screening is shown (Table 3).

[0104] Table 3 Indels that passed the Bonferroni correction (BONF) significance threshold based on GWAS screening (P < 0.05)

[0105] SNP F_A A1 F_U CHISQ A2 OR P CTGCAGGCCGATG 3 ri-3-47617128 C 0.512 0.1509 AACTAAGTCAGTC 36.14 1.84E-09 5.903 3 ri-3-47823327 A 0.3415 0.7019 CTGT 33.12 8.65E-09 0.2202 3 ri-3-47458918 CGT 0.358 0.717 C 33.02 9.10E-09 0.2201 3 ri-3-47460264 Gender 0.5904 0.2453 C 31.06 2.51E-08 4.434

[0106] Note: CHR: Chromosome, Chromosome number; SNP: SNP Identifier, Variant site ID; A1 : Allele 1, Effect Allele (Target Allele), Direction of detecting effect in association analysis (usually the minor allele or target phenotype-associated allele); F_A: Frequency in Cases, Frequency of A1 in cases (e.g. male) Allele frequency of A1 carried by male population; F_U: Frequency in Controls, Frequency of A1 in controls (e.g. female); A2: Allele 2, Reference Allele (Baseline Allele); CHISQ: Chi-square Statistic, Chi-square test statistic, Measure the size of genotype frequency difference between male and female populations (the larger the value, the more significant); P: P-value, Unadjusted association significance P-value; OR: Odds Ratio, Odds ratio (effect strength), OR = (F_A / (1-F_A)) / (F_U / (1-F_U)) (OR > 1 : A1 increases the probability of male).

[0107] Example 5 Molecular marker verification

[0108] 1. DNA extraction (fin tissue)

[0109] Reagents and equipment:

[0110] Reagent kit: TIANGEN marine animal genomic DNA extraction kit (DP324);

[0111] Tissue crusher: Tissuelyser LT (QIAGEN).

[0112] Steps:

[0113] Sample processing:

[0114] Take fin tissue ≤20 mg (ethanol fixed), cut with sterile blade and add 180 μL GA buffer + 20 μL Proteinase K lysis: 56°C water bath oscillation digestion for 3 hours (2 times for ordinary animal tissue);

[0115] DNA purification:

[0116] Add 200 μL GB buffer, incubate at 70°C for 10 min, add 200 μL anhydrous ethanol, transfer to the adsorption column, centrifuge at 12,000 rpm x 30 s (repeat 2 times).

[0117] Elution:

[0118] Add 60 μL preheated (65°C) ultrapure water for elution;

[0119] Output: concentration ≥ 25 ng / μL, OD260 / 280 = 1.82 ± 0.05.

[0120] 2. Primer design and synthesis

[0121] Label information:

[0122] Name: ri-3-47617128;

[0123] Location: Chr3: 47617128-47617139;

[0124] Variation: 12 bp deletion (male specific).

[0125] Primer design:

[0126] Forward primer: 5'-CCACCACTTCATTCCTGGACA-3'

[0127] Reverse primer: 5'-CTCATTCCTCTGCTCCCTACAA-3'

[0128] Product length:

[0129] - Wild type: 131 bp;

[0130] - Deletion type: 131 bp-12 bp = 119 bp.

[0131] Synthesis specifications:

[0132] Synthesis company: Sheng Wu Biotechnology

[0133] Purity: PAGE purification

[0134] Resuspension concentration: 100 μM (stock solution), working solution diluted to 10 μM;

[0135] The PCR reaction system used was: 2 × Taq MasterMix 10 μL, 0.5 μL of upstream and downstream primers with a concentration of 10 μM, 50 ng of genomic DNA (2 μL), ddH2O (6.4 μL) to 20 μL.

[0136] The PCR reaction program used was: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 60 °C annealing for 30 s, 72 °C extension for 9 s, 35 cycles, 72 °C extension for 5 min.

[0137] 3. Electrophoresis detection:

[0138] Gel concentration: 3% agarose gel (containing 0.5 × GelRed;

[0139] Electrophoresis conditions: 100 V × 1 h;

[0140] Loading amount: 5 μL PCR product;

[0141] The results are shown in Table 4:

[0142] Table 4: Results of electrophoresis detection

[0143] Number of bands Fragment size Female 1 band 131 bp Male 2 bands 131 bp + 119 bp Figure 6

[0144] Note: Ladder H2 DNA Marker (bands: 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1031 bp) was used.

[0145] 4. Sample verification design

[0146] The gender of the samples was identified by dissection and histology (see Example 2 for specific methods), as shown in Table 5 below.

[0147] Table 5: Gender of samples identified by dissection and histology

[0148]

[0149] 5. Typing results

[0150] Typical results of electrophoresis of some samples are shown in Figure 6 It can be seen from ​ that the 131 bp fragment exists only in the samples of phenotypic female, and both 131 bp and 119 bp fragments exist in the samples of phenotypic male. That is, female: single band (131 bp); male: double bands (131 bp and 119 bp).

[0151] 6. The accuracy statistics are shown in Table 6 below:

[0152] Table 6: Accuracy statistics of the method of the present application

[0153]

[0154] Conclusion: The primers designed using the ri-3-47617128 marker can achieve 100% accuracy in gender identification, and are applicable to wild Niaosou populations, Min-Yue-Dong populations, and domesticated Min-Yue-Dong populations. The entire detection can be completed within 4 hours, the cost of a single sample is less than 3 yuan, and it is suitable for large-scale screening.

[0155] The embodiments of the present application have been described in detail, but the present application is not limited to the described embodiments. Various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and still fall within the scope of the present application.

Claims

1. An indel marker for identifying the sex of large yellow croaker from different habitats, characterized in that, The indel marker is a 12bp insertion / deletion nucleotide sequence located at position 47617128 on chromosome 3 of the large yellow croaker, as shown in SEQ ID NO.

1. Female individuals have the sequence shown in SEQ ID NO.1, while male individuals lack the sequence shown in SEQ ID NO.

1.

2. A primer for amplifying the indel label of claim 1, characterized in that, The primers include an upstream primer and a downstream primer, the sequence of which is shown in SEQ ID NO.2 and the sequence of which is shown in SEQ ID NO.

3.

3. A kit for identifying the sex of large yellow croaker from different habitats, characterized in that: The kit includes the primers described in claim 2.

4. A method for identifying the sex of large yellow croaker from different habitats, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA of the sample to be tested as a template, perform PCR amplification using the primers described in claim 2 or the kit described in claim 3; (3) The sex of the large yellow croaker was determined based on the electrophoresis image. Individuals with only the 131bp fragment were female large yellow croakers, while individuals with both the 131bp and 119bp fragments were male large yellow croakers.

5. The method according to claim 4, characterized in that, In step (2), the PCR amplification process used the following PCR reaction system: 10 μL of 2×Taq MasterMix, 0.5 μL each of 10 μM upstream and downstream primers, 50 ng of genomic DNA, and ddH2O to 20 μL.

6. The method according to claim 4, characterized in that, In step (2), the PCR amplification process used was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 9 s, 35 cycles, and 72℃ extension for 5 min.

7. The application of the primers of claim 2 or the kit of claim 3 in identifying the sex of large yellow croaker in different habitats.

8. The application of the method according to any one of claims 4-6 in identifying the sex of large yellow croaker from different habitats.

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